Influenza‐associated respiratory illness among five cohorts of pregnant women and their young infants (0–6 months), Bangladesh, 2013–2017

Abstract Background Pregnant women with their infants are considered at higher risk for influenza‐associated complications, and the World Health Organization (WHO) recommends influenza vaccination during pregnancy to protect them, including their infants (0–6 months). There are limited data on the influenza burden among pregnant women and their infants (0–6 months), and there are no routine influenza vaccinations in Bangladesh. Methods Five annual cohorts (2013–2017) of pregnant women were enrolled from the eight sub‐districts of Bangladesh before the influenza season (May–September); they were contacted weekly to identify new onset of influenza‐like illness (ILI) (subjective or measured fever and cough) and acute respiratory illness (ARI) (at least two of these symptoms: cough, rhinorrhea, or difficulty in breathing) among their infants from birth to 6 months of age. We collected nasopharyngeal swabs from ILI and ARI cases, tested by real‐time reverse transcription polymerase chain reaction (rRT‐PCR) for influenza virus (including types and subtypes) and estimated influenza incidence (95% CI)/10000 pregnant women‐months or infant‐months, respectively. Results We enrolled 9020 pregnant women, followed for 26,709 pregnancy‐months, and detected 1241 ILI episodes. We also followed 8963 infants for 51,518 infant‐months and identified 5116 ARI episodes. Influenza positivity was 23% for ILI and 3% for ARI cases. The overall incidence (2013–2017) of influenza among pregnant women was 158.5/10000 pregnant women‐months (95% CI: 141.4–177.6) and that among infants was 21.9/10000 infant‐months (95% CI: 18.2–26.5). Conclusions Although the data was collected more than 5 years ago, as the only baseline data, our findings illustrate evidence of influenza burden among pregnant women and infants (0–6 months), which may support preventive policy decisions in Bangladesh.


| INTRODUCTION
Epidemiological studies have shown that pregnant women are at higher risk for influenza-associated hospitalizations during annual seasonal epidemics 1 and have an increased hospitalization rate for respiratory diseases during the annual influenza season than non-pregnant women. 2 In light of clear evidence of the risks associated with influenza virus infection among pregnant women, the World Health Organization's (WHO) strategic advisory group of experts (SAGE) working group considers pregnant women as a high priority risk group and has recommended influenza vaccination. 3 Postpartum women and infants <6 months are also at increased risk of severe influenza virus infections. 4,5 Because infants <6 months of age cannot be vaccinated against influenza, the risk of severe illness due to influenza virus infection remains high. 6 A study from Mongolia from 2013 to 2015 showed that 7.4% of infants <6 months of age had influenza virus infections. 7 A previous study in Bangladesh in 2009 showed a high incidence (6/100 child years) of influenza virus infection among infants <6 months of age. 8 Annual influenza vaccination has been an important measure of preventing and controlling influenza virus infections. 9 Influenza vaccination during pregnancy was reported to reduce the incidence of influenza illnesses and hospitalizations among infants <6 months of age. 10,11 However, in most low-income countries, including Bangladesh, the influenza vaccine is not part of routine immunization practices, 12 and pregnant women are unlikely to be vaccinated.
There are limited data on the burden of laboratory-confirmed seasonal influenza illnesses among pregnant women and their young infants in Bangladesh. 13 These data are helpful for policymakers to consider future control and prevention strategies against influenza like surveillance, non-pharmaceutical interventions, and vaccination.
This study estimated the incidence of influenza virus infection among pregnant women and their infants <6 months of age during the five influenza seasons in four administrative districts of Bangladesh.

| Study population and setting
From 2013 to 2017, five cohorts of pregnant women of any gestational age in urban and peri-urban areas of Cumilla, Bogura, Kishoreganj, and Barishal districts of Bangladesh were enrolled, followed until delivery.
Then, their infants were followed from birth to 6 months of age to detect influenza virus infections ( Figure 1).

| Study design and sampling strategy
We conducted a prospective cohort study that enrolled pregnant women annually during March and April before the start of the influenza season, typically from May to September. 14 To formulate the enrollment strategy, we reviewed information about the use of antenatal care (ANC) services in the four districts selected. Five pregnant women visited an ANC facility each day at the eight local sub-district health facilities for ANC services. Therefore, we considered 2 months of enrolment before the start of the influenza season, targeting to enroll 2080 (equivalent to five pregnant women times eight subdistrict health facilities times 26 days per month for 2 months) pregnant women from eight sub-district health facilities. We also sought to enroll another 500 pregnant women from the Government Family Planning Department's register list. We assumed 15% of refusal from the women visiting the sub-district health facility and 10% loss of follow-up of all enrolled women during the study period and F I G U R E 1 Study sites in four districts of Bangladesh, 2013Bangladesh, -2017 accounted for those assumptions. Considering these assumptions, we formulated our sampling strategy of 2000 pregnant women per cohort per year, and it would yield an influenza incidence of laboratoryconfirmed infection among symptomatic pregnant women in Bangladesh.
For the first cohort, the enrollment of 2000 pregnant women was delayed and occurred from May to June 2013, after the IRB approval.
We recruited the subsequent cohorts (2014-2017) using the Government Family Planning Department's register from the catchment area of eight local (sub-district) health complexes. Leveraging this register, we sought to enroll 2000 pregnant women by April of each year. As there was no routine influenza vaccination in Bangladesh, 12 all our study participants were likely to be unvaccinated. We then followed up on enrolled cohorts of pregnant women from 2013 to 2017 through the end of pregnancy, irrespective of live birth. We followed infants born to these pregnant women from birth to 6 months.
Regardless of the pregnancy's gestational age, we requested all pregnant women to participate and enroll. Written informed consent to participate in the study was obtained from the participant or the infant's mother before enrolling, data collection, or specimen collection. The study was approved by the icddr,b institutional review board (IRB) before enrolling participants. Centers for Disease Control and Prevention (CDC)'s Human Research Protection Office has reviewed and approved the reliance on icddr,b's IRB.

| Case definitions, participant follow-up, data collection, and biological specimen testing
We adopted case definitions from the influenza-like illness (ILI) case definition of the WHO. 15 For pregnant women, a case definition of ILI was defined as subjective or measured fever and cough in the previous 7 days. For infants, a case definition of acute respiratory illness (ARI) was defined as the new onset of at least two signs or symptoms: cough, rhinorrhea, or difficulty breathing during the last 7 days.
After enrollment, we collected socio-demographic and socioeconomic information using a structured questionnaire. Gestational age was measured using the date of the last menstrual period (LMP). Due to participants' potential recall bias about the LMP, we piloted the feasibility of use of a portable ultrasound machine in community settings. With limited resources, in 2016 and 2017, we sought to perform portable transabdominal ultrasounds among a subset of pregnant women at <24 weeks gestation as determined previously by LMP. 16 Therefore, with resources available, this ultrasound could be performed in a community setting to have more accurate data.
To capture infant birth weight and enroll infants, the study staff conducted a household visit within 3 days following birth. From 2013 to 2015, the study staff measured birth weight with digital household scales with measurement precision to the nearest 100 g using a standard operating procedure. For the 2016-2017 cohorts, study staff registered the birth weight using baby scales (beurer BY 80), which measured weight to the nearest 5 g. These baby scales were available locally and had automatic and manual hold functions that provided more accurate birth weight measurements. For births in medical facilities, we obtained infant birth weight from birth certificates.
Birth weight of <2500 g was considered a low birth weight (LBW). 17 We defined stillbirth as a fetal death occurring after ≥28 weeks of gestation. 18 Follow up began coinciding with the influenza season, and it was conducted once weekly with mobile phone calls or home visits if the participant was unreachable by phone. Study team had to ensure that they have contacted respondents either by phone or home visit every work day so that there were no loss to follow up unless the participant refused to continue participating in the study. Infants were followed once per week from birth to 6 months of age. Each week, we asked pregnant women if they experienced a new ILI and met the ILI case definition; a household visit was scheduled for the same day to collect detailed illness information and a nasopharyngeal (NP) swab.
We considered a repeat ILI episode if the symptom onset dates of the episodes were more than 14 days apart. However, we did not consider any definite period of resolved symptoms.
Similarly, for infants, each week, we asked mothers if their infants had experienced any new respiratory signs or symptoms since the last follow-up. We asked the mothers if their infant suffered an ARI and met the ARI case definition; we scheduled a household visit on the same day to collect detailed illness information and an NP swab. We considered a repeat ARI episode if symptom onset dates between the episodes were more than 14 days apart.
The NP swabs were stored in a viral transport media, kept inside nitrogen dry-shippers, and transported to the virology laboratory at icddr,b in Dhaka (capital city) every 2 weeks for influenza virus testing (both type and subtype testing for A and lineage testing for B) by realtime reverse transcription polymerase chain reaction (rRT-PCR) using primers and probes from US CDC.
We considered a loss to follow up if a respondent withdrew consent or migrated out of the study catchment area, age verification of a pregnant woman of ≥18 years was not possible, or the termination of pregnancy by any means before the commencement of follow-up.

| Data analysis
We summarized demographic information for pregnant women and infants using descriptive statistics. We used principal component analysis to calculate the participant wealth index using data on economic statuses, durable asset ownership, access to utilities and infrastructure, and housing characteristics. We analyzed additional demographic and pregnancy characteristics such as parity and gestational age for their association with having an influenza virus infection. We compared gestational age obtained by LMP and ultrasound of the subset of the same respondents and used a paired t-test to analyze differences in results obtained by the two methods.
We described circulating influenza viruses by type and subtype, reported ILI and influenza symptoms, the clinical course of illness episodes, and the number of individuals with repeat ILI/ARI episodes.
We summarized the information regarding illness symptoms for pregnant women by ILI episodes and influenza virus infection. Similarly, we summarized illness information for infants by ARI episode and influenza virus illness.
We analyzed influenza virus illness incidence among pregnant women and infants <6 months of age by influenza subtype. We calcu-  (Table 3). We found that 98% of illness episodes among pregnant women were in the third trimester of pregnancy. The

| DISCUSSION
Our study was a resource-intense effort to document the incidence of influenza among pregnant women for the first time in Bangladesh. We    to non-hospitalized cases. 28 Findings of no hospitalization and no adverse outcomes are suggestive that influenza was not severe. However, these were not our study objectives, and the sample size was not powered enough to determine hospitalizations and adverse outcomes.
As there was concern about the accuracy of measuring gestational age using the date of the LMP because of potential recall bias and the difficulty of predicting gestational age by menstrual history, 29 we explored the feasibility of using portable ultrasound machines. We observed a statistically significant difference in gestational age deter- were born towards the end of the typical influenza circulation period.